Conformational changes at the tetramerization site of erythroid α-spectrin upon binding β-spectrin:: A spin label EPR study

We used cysteine scanning, isothermal titration calorimetry (ITC) and spin label EPR methods to study the two regions that flank the partial domain Helix C' of the N-terminal end of alpha-spectrin (residues 14-20 and residues 44-54) in the absence and presence of a model protein of the beta-spectrin C-terminal end. In the absence of beta-spectrin, residues 14-20 and 46-52 were known to be unstructured. The EPR spectral values of the inverse line width (Delta H-1) and of the width between the low field peak and the central peak (alpha Z) of residues in part of the first unstructured region (residues 17-20) and of most residues in the second unstructured junction region (residues 46-52) changed dramatically upon association with beta-spectrin, suggesting that the two regions undergo a conformational change, becoming more rigid and likely becoming helical. ITC results showed that three of the seven residues in the junction region (residues 46-52) were very important in its association with beta-spectrin, in the following order: L49 > G46 > K48. In general, our results suggest that any mutations that affect the propensity of helical formation in the region spanning residues 17-52 in alpha-spectrin, or that affect hydrophobic clustering and/or salt-bridge stabilization of the bundled helices, would affect spectrin tetramer formation, and may lead to blood disorders.